The complex molybdohemoflavoprotein, NADH:nitrate reductase (NR; E.C. 1.6.6.1). catalyzes the reduction of nitrate to nitrite using NADH as the physiological electron donor, a reaction that represents the regulated and rate-limiting step in the pathway of nitrogen assimilation in eukaryotes. The dimeric enzyme exhibits a tripartite structure with individual subunits comprising functional domains that contain Mo-pterin, cytochrome b557 and FAD prosthetic groups in a 1:1:1 stoichiometry with FAD as the site of electron ingress and Mo-pterin as the egress site for the conversion of nitrate to nitrite. The overall goal of the proposed research is to identify and characterize molecular and physical determinants that regulate the catalytic efficiency of NR, which represents an excellent model for investigating electron transfer reactions in complex, multi-center proteins containing diverse prosthetic groups and which shares a number of common structural and mechanistic features with a variety of other flavin-, heme- and Mo-containing proteins including cytochrome b5 reductase, ferredoxin:NADP+ reductase, cytochrome b5 and sulfite oxidase. We pioneered a reductionist approach and have successfully developed heterologous expression systems for the individual, functional Mo-pterin, heme and FAD domains as well as the Mo-pterin/heme and heme/FAD fusion domain, to probe factors influencing cofactor redox potentials and the regulation of FAD-heme and heme-Mo-pterin energy transduction. The proposed research will focus on integrating site-directed mutagenesis and extensive spectroscopic, thermodynamic and structural analyses to define the roles of selected amino acid residues in the various NR domains in controlling cofactor redox potentials together with examining the effects of relative cofactor redox potential modulation on the efficiency of energy transduction within NR. Specific motifs including the RXY-T sXXsN and "CGXXXM" sequences involved in FAD- and NADH-binding, the role of the Mo-heme "linker" region in controlling the heme potential and the role of the "CAG" Mo-pterin signature will be examined. These studies will enhance our understanding of important structure-function relationships in NR and other related metalloflavoproteins.